Recent investigations have recognized that second messenger responsive targets are sequestered within discrete domains of the cell. This view of the intracellular world has led to the recognition of broad groups of scaffolding proteins, which sequester both transducing enzymes and substrates in defined locations within cells. The best-characterized and most extensive groups of these scaffolding proteins are the A-kinase anchoring proteins (AKAPs), which bind a dimer of regulatory subunits of Type Il A-kinase. We have focused our investigations over the past decade on AKAPs associated with gastric parietal cell function. These studies have led to the identification of both ezrin and AKAP35O as AKAPs with important scaffolding functions of more general importance in epithelial cells. The present investigations center on the function of the multiply spliced AKAP35O family. In addition to Type II A-kinase, these proteins also scaffold protein phosphatases 1 and 2a as well as protein kinase N (Rho kinase) and protein kinase C-epsilon. Recently in polarized HCA-7 colon carcinoma cells and parietal cells, we have demonstrated that a major splice variant AKAP35OA is specifically associated with the Golgi apparatus. We have hypothesized that AKAP35OA at the Golgi apparatus serves as a multi-functional scaffolding system for the anchoring of critical regulators of polarized epithelial function. Indeed, we have recently identified two novel families of AKAP35O interacting proteins, Chloride Intracellular Channels (CLICs) and CIP4ICIP5, putative cross linkers of the actin and microtubule cytoskeletons, which associate with AKAP35OA at the Golgi apparatus. We will seek to identify and characterize common and specific functions of interacting proteins associated with the large multiprotein AKAP35O scaffolded complexes. To accomplish these goals we will pursue three specific aims: First, we will characterize the functional association of CIP4 and CIP5 with AKAP35OA at the Golgi apparatus and their roles in regulating protein trafficking. Second, we will investigate the structural and functional association of AKAP35OA with CLIC5B anchoring at the Golgi apparatus. Third, we will isolate and identify the components of the large non-centrosomal AKAP5O scaffolded complexes in gastric parietal cells and HCA-7 colon carcinoma cells. These studies will allow focused investigation of the spectrum of AKAP35O scaffolding complexes that may regulate intra-Golgi and post-Golgi trafficking in polarized epithelial cells.